Diagnostic utility of droplet digital PCR for HIV reservoir quantification
Identifieur interne : 000074 ( Pmc/Corpus ); précédent : 000073; suivant : 000075Diagnostic utility of droplet digital PCR for HIV reservoir quantification
Auteurs : Wim Trypsteen ; Maja Kiselinova ; Linos Vandekerckhove ; Ward De SpiegelaereSource :
- Journal of Virus Eradication [ 2055-6640 ] ; ????.
Abstract
Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts.
Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility.
We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination).
In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved.
Url:
PubMed: 27482456
PubMed Central: 4967968
Links to Exploration step
PMC:4967968Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Diagnostic utility of droplet digital PCR for HIV reservoir quantification</title><author><name sortKey="Trypsteen, Wim" sort="Trypsteen, Wim" uniqKey="Trypsteen W" first="Wim" last="Trypsteen">Wim Trypsteen</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="Kiselinova, Maja" sort="Kiselinova, Maja" uniqKey="Kiselinova M" first="Maja" last="Kiselinova">Maja Kiselinova</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="Vandekerckhove, Linos" sort="Vandekerckhove, Linos" uniqKey="Vandekerckhove L" first="Linos" last="Vandekerckhove">Linos Vandekerckhove</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="De Spiegelaere, Ward" sort="De Spiegelaere, Ward" uniqKey="De Spiegelaere W" first="Ward" last="De Spiegelaere">Ward De Spiegelaere</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27482456</idno><idno type="pmc">4967968</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967968</idno><idno type="RBID">PMC:4967968</idno><date when="????">????</date><idno type="wicri:Area/Pmc/Corpus">000074</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Diagnostic utility of droplet digital PCR for HIV reservoir quantification</title><author><name sortKey="Trypsteen, Wim" sort="Trypsteen, Wim" uniqKey="Trypsteen W" first="Wim" last="Trypsteen">Wim Trypsteen</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="Kiselinova, Maja" sort="Kiselinova, Maja" uniqKey="Kiselinova M" first="Maja" last="Kiselinova">Maja Kiselinova</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="Vandekerckhove, Linos" sort="Vandekerckhove, Linos" uniqKey="Vandekerckhove L" first="Linos" last="Vandekerckhove">Linos Vandekerckhove</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author><author><name sortKey="De Spiegelaere, Ward" sort="De Spiegelaere, Ward" uniqKey="De Spiegelaere W" first="Ward" last="De Spiegelaere">Ward De Spiegelaere</name><affiliation><nlm:aff id="jve1-aff-0001"><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Virus Eradication</title><idno type="ISSN">2055-6640</idno><idno type="eISSN">2055-6659</idno><imprint><date when="????">????</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Abstract</title><p id="jve1-para-0001">Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts.</p><p id="jve1-para-0002">Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility.</p><p id="jve1-para-0003">We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination).</p><p id="jve1-para-0004">In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Huggett, Jf" uniqKey="Huggett J">JF Huggett</name></author><author><name sortKey="Cowen, S" uniqKey="Cowen S">S Cowen</name></author><author><name sortKey="Foy, Ca" uniqKey="Foy C">CA Foy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hindson, Cm" uniqKey="Hindson C">CM Hindson</name></author><author><name sortKey="Chevillet, Jr" uniqKey="Chevillet J">JR Chevillet</name></author><author><name sortKey="Briggs, Ha" uniqKey="Briggs H">HA Briggs</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sedlak, Rh" uniqKey="Sedlak R">RH Sedlak</name></author><author><name sortKey="Jerome, Kr" uniqKey="Jerome K">KR Jerome</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Simmonds, P" uniqKey="Simmonds P">P Simmonds</name></author><author><name sortKey="Balfe, P" uniqKey="Balfe P">P Balfe</name></author><author><name sortKey="Peutherer, Jf" uniqKey="Peutherer J">JF Peutherer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hindson, Bj" uniqKey="Hindson B">BJ Hindson</name></author><author><name sortKey="Ness, Kd" uniqKey="Ness K">KD Ness</name></author><author><name sortKey="Masquelier, Da" uniqKey="Masquelier D">DA Masquelier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pinheiro, Lb" uniqKey="Pinheiro L">LB Pinheiro</name></author><author><name sortKey="Coleman, Va" uniqKey="Coleman V">VA Coleman</name></author><author><name sortKey="Hindson, Cm" uniqKey="Hindson C">CM Hindson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vogelstein, B" uniqKey="Vogelstein B">B Vogelstein</name></author><author><name sortKey="Kinzler, Kw" uniqKey="Kinzler K">KW Kinzler</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Morley, Aa" uniqKey="Morley A">AA Morley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taly, V" uniqKey="Taly V">V Taly</name></author><author><name sortKey="Pekin, D" uniqKey="Pekin D">D Pekin</name></author><author><name sortKey="El Abed, A" uniqKey="El Abed A">A El Abed</name></author><author><name sortKey="Laurent Puig, P" uniqKey="Laurent Puig P">P Laurent-Puig</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Strain, Mc" uniqKey="Strain M">MC Strain</name></author><author><name sortKey="Richman, Dd" uniqKey="Richman D">DD Richman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Theodorsson, E" uniqKey="Theodorsson E">E Theodorsson</name></author><author><name sortKey="Magnusson, B" uniqKey="Magnusson B">B Magnusson</name></author><author><name sortKey="Leito, I" uniqKey="Leito I">I Leito</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Westgard, Jo" uniqKey="Westgard J">JO Westgard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Armbruster, Da" uniqKey="Armbruster D">DA Armbruster</name></author><author><name sortKey="Pry, T" uniqKey="Pry T">T Pry</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Henrich, Tj" uniqKey="Henrich T">TJ Henrich</name></author><author><name sortKey="Gallien, S" uniqKey="Gallien S">S Gallien</name></author><author><name sortKey="Li, Jz" uniqKey="Li J">JZ Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eriksson, S" uniqKey="Eriksson S">S Eriksson</name></author><author><name sortKey="Graf, Eh" uniqKey="Graf E">EH Graf</name></author><author><name sortKey="Dahl, V" uniqKey="Dahl V">V Dahl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Strain, Mc" uniqKey="Strain M">MC Strain</name></author><author><name sortKey="Lada, Sm" uniqKey="Lada S">SM Lada</name></author><author><name sortKey="Luong, T" uniqKey="Luong T">T Luong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hatano, H" uniqKey="Hatano H">H Hatano</name></author><author><name sortKey="Strain, Mc" uniqKey="Strain M">MC Strain</name></author><author><name sortKey="Scherzer, R" uniqKey="Scherzer R">R Scherzer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yukl, Sa" uniqKey="Yukl S">SA Yukl</name></author><author><name sortKey="Boritz, E" uniqKey="Boritz E">E Boritz</name></author><author><name sortKey="Busch, M" uniqKey="Busch M">M Busch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kiselinova, M" uniqKey="Kiselinova M">M Kiselinova</name></author><author><name sortKey="Pasternak, Ao" uniqKey="Pasternak A">AO Pasternak</name></author><author><name sortKey="De Spiegelaere, W" uniqKey="De Spiegelaere W">W De Spiegelaere</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rasmussen, Ta" uniqKey="Rasmussen T">TA Rasmussen</name></author><author><name sortKey="Tolstrup, M" uniqKey="Tolstrup M">M Tolstrup</name></author><author><name sortKey="Brinkmann, Cr" uniqKey="Brinkmann C">CR Brinkmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruelle, J" uniqKey="Ruelle J">J Ruelle</name></author><author><name sortKey="Yfantis, V" uniqKey="Yfantis V">V Yfantis</name></author><author><name sortKey="Duquenne, A" uniqKey="Duquenne A">A Duquenne</name></author><author><name sortKey="Goubau, P" uniqKey="Goubau P">P Goubau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kiselinova, M" uniqKey="Kiselinova M">M Kiselinova</name></author><author><name sortKey="Anna, M" uniqKey="Anna M">M Anna</name></author><author><name sortKey="Malatinkova, E" uniqKey="Malatinkova E">E Malatinkova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kiselinova, M" uniqKey="Kiselinova M">M Kiselinova</name></author><author><name sortKey="Geretti, Am" uniqKey="Geretti A">AM Geretti</name></author><author><name sortKey="Malatinkova, E" uniqKey="Malatinkova E">E Malatinkova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Malatinkova, E" uniqKey="Malatinkova E">E Malatinkova</name></author><author><name sortKey="De Spiegelaere, W" uniqKey="De Spiegelaere W">W De Spiegelaere</name></author><author><name sortKey="Bonczkowski, P" uniqKey="Bonczkowski P">P Bonczkowski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pallikkuth, S" uniqKey="Pallikkuth S">S Pallikkuth</name></author><author><name sortKey="Sharkey, M" uniqKey="Sharkey M">M Sharkey</name></author><author><name sortKey="Babic, Dz" uniqKey="Babic D">DZ Babic</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sogaard, Os" uniqKey="Sogaard O">OS Sogaard</name></author><author><name sortKey="Graversen, Me" uniqKey="Graversen M">ME Graversen</name></author><author><name sortKey="Leth, S" uniqKey="Leth S">S Leth</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruggiero, A" uniqKey="Ruggiero A">A Ruggiero</name></author><author><name sortKey="De Spiegelaere, W" uniqKey="De Spiegelaere W">W De Spiegelaere</name></author><author><name sortKey="Cozzi Lepri, A" uniqKey="Cozzi Lepri A">A Cozzi-Lepri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bosman, Kj" uniqKey="Bosman K">KJ Bosman</name></author><author><name sortKey="Nijhuis, M" uniqKey="Nijhuis M">M Nijhuis</name></author><author><name sortKey="Ham, Pm" uniqKey="Ham P">PM Ham</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="King, Cc" uniqKey="King C">CC King</name></author><author><name sortKey="Kourtis, Ap" uniqKey="Kourtis A">AP Kourtis</name></author><author><name sortKey="Persaud, D" uniqKey="Persaud D">D Persaud</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Oliveira, Mf" uniqKey="Oliveira M">MF Oliveira</name></author><author><name sortKey="Gianella, S" uniqKey="Gianella S">S Gianella</name></author><author><name sortKey="Letendre, S" uniqKey="Letendre S">S Letendre</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kiselinova, M" uniqKey="Kiselinova M">M Kiselinova</name></author><author><name sortKey="De Spiegelaere, W" uniqKey="De Spiegelaere W">W De Spiegelaere</name></author><author><name sortKey="Buzon, Mj" uniqKey="Buzon M">MJ Buzon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hutter, G" uniqKey="Hutter G">G Hutter</name></author><author><name sortKey="Nowak, D" uniqKey="Nowak D">D Nowak</name></author><author><name sortKey="Mossner, M" uniqKey="Mossner M">M Mossner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ho, Yc" uniqKey="Ho Y">YC Ho</name></author><author><name sortKey="Shan, L" uniqKey="Shan L">L Shan</name></author><author><name sortKey="Hosmane, Nn" uniqKey="Hosmane N">NN Hosmane</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jones, M" uniqKey="Jones M">M Jones</name></author><author><name sortKey="Williams, J" uniqKey="Williams J">J Williams</name></author><author><name sortKey="Gartner, K" uniqKey="Gartner K">K Gartner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dreo, T" uniqKey="Dreo T">T Dreo</name></author><author><name sortKey="Pirc, M" uniqKey="Pirc M">M Pirc</name></author><author><name sortKey="Ramsak, Z" uniqKey="Ramsak Z">Z Ramsak</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Trypsteen, W" uniqKey="Trypsteen W">W Trypsteen</name></author><author><name sortKey="Vynck, M" uniqKey="Vynck M">M Vynck</name></author><author><name sortKey="De Neve, J" uniqKey="De Neve J">J De Neve</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="editorial"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Virus Erad</journal-id><journal-id journal-id-type="iso-abbrev">J Virus Erad</journal-id><journal-id journal-id-type="publisher-id">JOURNAL OF VIRUS ERADICATION</journal-id><journal-title-group><journal-title>Journal of Virus Eradication</journal-title></journal-title-group><issn pub-type="ppub">2055-6640</issn><issn pub-type="epub">2055-6659</issn><publisher><publisher-name>Mediscript Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27482456</article-id><article-id pub-id-type="pmc">4967968</article-id><article-categories><subj-group subj-group-type="heading"><subject>Review</subject></subj-group></article-categories><title-group><article-title>Diagnostic utility of droplet digital PCR for HIV reservoir quantification</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Trypsteen</surname><given-names>Wim</given-names></name><xref ref-type="aff" rid="jve1-aff-0001"></xref></contrib><contrib contrib-type="author"><name><surname>Kiselinova</surname><given-names>Maja</given-names></name><xref ref-type="aff" rid="jve1-aff-0001"></xref></contrib><contrib contrib-type="author"><name><surname>Vandekerckhove</surname><given-names>Linos</given-names></name><xref ref-type="aff" rid="jve1-aff-0001"></xref><xref ref-type="corresp" rid="an1">*</xref></contrib><contrib contrib-type="author"><name><surname>De Spiegelaere</surname><given-names>Ward</given-names></name><xref ref-type="aff" rid="jve1-aff-0001"></xref></contrib><aff id="jve1-aff-0001"><label></label><institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,<institution>Ghent University</institution>,<country>Belgium</country></aff></contrib-group><author-notes><corresp id="an1"><label>*</label>Corresponding author: Linos Vandekerckhove,
<institution>HIV Translational Research Unit, Department of Internal Medicine</institution>,
<addr-line>De Pintelaan 185, Medical Research Building 2</addr-line>,
<institution>Ghent University</institution>,
<addr-line>9000</addr-line><addr-line>Ghent</addr-line>,
<country>Belgium</country>Email:
<email>linos.vandekerckhove@ugent.be</email></corresp></author-notes><pub-date date-type="collection" publication-format="electronic"><month>7</month><year>2016</year></pub-date><pub-date date-type="pub" publication-format="electronic"><day>1</day><month>7</month><year>2016</year></pub-date><volume>2</volume><issue>3</issue><fpage>162</fpage><lpage>169</lpage><permissions><copyright-statement>© 2016 The Authors. <italic>Journal of Virus Eradication</italic> published by Mediscript Ltd</copyright-statement><license license-type="open-access"><license-p>This is an open access article published under the terms of a Creative Commons License.</license-p></license></permissions><abstract id="jve1-abs-0001"><title>Abstract</title><p id="jve1-para-0001">Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts.</p><p id="jve1-para-0002">Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility.</p><p id="jve1-para-0003">We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination).</p><p id="jve1-para-0004">In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved.</p></abstract><kwd-group kwd-group-type="author"><title id="jve1-title-0002">Keywords</title><kwd>droplet digital PCR</kwd><kwd>digital PCR</kwd><kwd>quantitative real-time PCR</kwd><kwd>HIV quantification</kwd><kwd>virus detection</kwd></kwd-group></article-meta></front><body><sec id="jve1-sec-0001"><title id="jve1-title-0003">Introduction</title><p id="jve1-para-0005">In the last two decades, quantitative real-time PCR (qPCR) has become a standard tool in many molecular laboratories for the quantification of viral nucleic acids and rigorously implemented in accredited clinical testing <xref rid="jve1-bib-0001" ref-type="bibr">[1]</xref>. Despite its success, qPCR remains an indirect quantification method that relies on pre-validated standard curves, and is susceptible to small changes in reaction efficiency, which can substantially bias quantification due to the logarithmic nature of a PCR reaction. In this context, digital PCR (dPCR) provides an alternative type of technology that benefits from direct quantification without the need for a standard curve, and is associated with a better tolerance to small changes in PCR efficiency <xref rid="jve1-bib-0002" ref-type="bibr">[2,3]</xref>.</p><p id="jve1-para-0006">The concept of dPCR-based quantification, initially called single molecule PCR, was described before the invention of qPCR <xref rid="jve1-bib-0004" ref-type="bibr">[4]</xref>. When using dPCR, a sample is divided into multiple partitions, each representing an isolated end-point PCR reaction. In a sufficiently diluted sample (at a limiting dilution), the distribution of the template molecules within these partitions follows a Poisson distribution. Consequently, this distribution can be used to deduce the concentration of target nucleic acids in a sample from the frequency of positive to negative partitions in the dPCR <xref rid="jve1-bib-0005" ref-type="bibr">[5–7]</xref>.</p><p id="jve1-para-0007">Notwithstanding the straightforward principle of dPCR, the high costs and labour-intensive procedures to generate high numbers of PCR partitions had hampered a wider use of dPCR until recently <xref rid="jve1-bib-0008" ref-type="bibr">[8]</xref>. Thanks to technological developments in microfluidics, a series of digital PCR platforms has been developed that can generate high numbers of PCR partitions per sample at a reasonable price <xref rid="jve1-bib-0009" ref-type="bibr">[9]</xref>. However, as with all new technologies, extensive optimisation and validation are required to assess whether the newer technology offers an improved platform for nucleic acid quantification and is able to provide additional benefits.</p><p id="jve1-para-0008">The advantages of such technology are especially promising in the setting of HIV reservoir and cure research because the quantification of HIV reservoirs by PCR becomes challenging when addressing small reservoir sizes and subtle changes in reservoir dynamics <xref rid="jve1-bib-0010" ref-type="bibr">[10]</xref>. Therefore, this review explores and compares dPCR with qPCR applications in the field of HIV research in particular. It also focuses on droplet digital PCR (ddPCR), as this is currently the most frequently used dPCR platform (Figure <xref ref-type="fig" rid="jve-2-162-g001">1</xref>).</p><fig id="jve-2-162-g001" orientation="portrait" position="float"><label>Figure 1.</label><caption><p id="jve1-para-0009">Reported use of different digital PCR platforms over time in published articles from November 2011 until March 2016. The absolute numbers (a, c) and ratios in percentage (b, d) are depicted for ddPCR <italic>vs</italic> non-ddPCR usage in a general dPCR context (a, b) and in an HIV-specific context (c, d)</p></caption><graphic xlink:href="jve-2-162-g001"></graphic></fig></sec><sec id="jve1-sec-0002"><title id="jve1-title-0004">Materials and methods</title><sec id="jve1-sec-0003"><title id="jve1-title-0005">Systematic literature screen</title><p id="jve1-para-0010">We performed a systematic literature search of ddPCR in the context of HIV quantification using the PubMed (
<uri xlink:href="http://www.ncbi.nlm.nih.gov/pubmed">www.ncbi.nlm.nih.gov/pubmed</uri>) and ISI Web of Science databases. The following search terms were used to construct the initial dataset of articles: ‘HIV’, ‘polymerase chain reaction’ and ‘PCR’. Next, only research articles and English-language manuscripts were considered for the period between November 2011 (first report on ddPCR) and March 2016. In addition, the use of ddPCR was also searched for in clinical trials related to HIV intervention studies with published articles (on
<uri xlink:href="http://clinicaltrials.gov">clinicaltrials.gov</uri>). Therefore, the following search term was used: ‘HIV reservoir’, with additional criteria: interventional studies received from November 2011 until March 2016. Subsequently, all articles were pooled together, duplicates were removed and text mining was performed within the available full text articles using the following search terms: ‘droplet digital PCR’ and ‘ddPCR’ (<xref rid="jve1-title-0025">Appendix 1</xref>). The remaining set of articles was manually evaluated with the following exclusion criteria: (1) ddPCR was used for the quantification of other assays than HIV; (2) only HIV qPCR was performed; (3) review articles; (4) descriptive data analysis method; and (5) HIV quantification was not performed on patient samples (<xref rid="jve1-title-0026">Appendix 2</xref>).</p></sec><sec id="jve1-sec-0004"><title id="jve1-title-0006">Evaluation criteria for the comparison of ddPCR and qPCR</title><p id="jve1-para-0011">The ddPCR and qPCR platforms were compared in terms of their different technical aspects, when data was available (Table <xref ref-type="table" rid="jve1-tbl-0001">1</xref>).</p><table-wrap id="jve1-tbl-0001" orientation="portrait" position="float"><label>Table 1.</label><caption><p id="jve1-para-0012">Overview of technical aspects used for comparison of ddPCR and qPCR</p></caption><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="left" span="1"></col></colgroup><thead><tr><th rowspan="1" colspan="1">Technical aspect</th><th rowspan="1" colspan="1">Definition</th></tr></thead><tbody><tr><td rowspan="1" colspan="1"><bold>Accuracy bias</bold></td><td rowspan="1" colspan="1">The closeness of a single measurement to the true value <xref rid="jve1-bib-0011" ref-type="bibr">[11,12]</xref><break></break>Difference between the mean of measurements and the true value <xref rid="jve1-bib-0011" ref-type="bibr">[11,12]</xref></td></tr><tr><td rowspan="1" colspan="1"><bold>Precision</bold></td><td rowspan="1" colspan="1">Closeness of agreement among replicate measurements on the same sample <xref rid="jve1-bib-0011" ref-type="bibr">[11,12]</xref></td></tr><tr><td rowspan="1" colspan="1"><bold>Reproducibility</bold></td><td rowspan="1" colspan="1">Variation for the same measurement process among different instruments, operators and over a longer period of time <xref rid="jve1-bib-0011" ref-type="bibr">[11]</xref></td></tr><tr><td rowspan="1" colspan="1"><bold>Sensitivity</bold><break></break><bold>Limit of detection</bold></td><td rowspan="1" colspan="1">The smallest concentration of a analyte that can be reliably measured by an analytical procedure <xref rid="jve1-bib-0013" ref-type="bibr">[13]</xref><break></break>Lowest analyte concentration likely to be reliably distinguished from the limit of blank<xref rid="jve1-bib-0013" ref-type="bibr">[13]</xref></td></tr><tr><td rowspan="1" colspan="1"><bold>Limit of blank</bold></td><td rowspan="1" colspan="1">Highest ‘apparent’ analyte concentration found in a blank sample <xref rid="jve1-bib-0013" ref-type="bibr">[13]</xref></td></tr></tbody></table></table-wrap></sec></sec><sec id="jve1-sec-0005"><title id="jve1-title-0007">Results</title><sec id="jve1-sec-0006"><title id="jve1-title-0008">HIV quantification assays: systematic literature search</title><p id="jve1-para-0013">The systematic literature search on the use of droplet digital PCR in the context of HIV quantification using patient samples resulted 19 articles that were included in this review (Figure <xref ref-type="fig" rid="jve-2-162-g002">2</xref>).</p><fig id="jve-2-162-g002" orientation="portrait" position="float"><label>Figure 2.</label><caption><p id="jve1-para-0014">Overview of systematic pipeline used for this review</p></caption><graphic xlink:href="jve-2-162-g002"></graphic></fig><p id="jve1-para-0015">HIV quantification assays can be divided into PCR- and non-PCR-based approaches and are focused on the different HIV forms/intermediates (Table <xref ref-type="table" rid="jve1-tbl-0002">2</xref>). Across all reports, we observed that ddPCR was used for the quantification of multiple HIV-1 and HIV-2 forms/intermediates: total HIV DNA, 2-LTR circles, HIV unspliced-RNA and HIV multiple-spliced RNA (Figure <xref ref-type="fig" rid="jve-2-162-g003">3</xref>). The majority of assays included HIV DNA-based applications (total HIV DNA and 2-LTR circles) and ddPCR was not only used for the quantification of integrated HIV DNA.</p><table-wrap id="jve1-tbl-0002" orientation="portrait" position="float"><label>Table 2.</label><caption><p id="jve1-para-0016">Overview of reference methods for HIV reservoir quantification</p></caption><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="left" span="1"></col></colgroup><thead><tr><th rowspan="1" colspan="1">HIV quantification method</th><th rowspan="1" colspan="1">What is measured</th></tr></thead><tbody><tr><td colspan="2" rowspan="1"><bold><italic>PCR-based methods</italic></bold></td></tr><tr><td rowspan="1" colspan="1">Total HIV DNA</td><td rowspan="1" colspan="1">Total pool of HIV DNA in the cell</td></tr><tr><td rowspan="1" colspan="1">Integrated HIV DNA</td><td rowspan="1" colspan="1">Integrated fraction of HIV DNA in the genome</td></tr><tr><td rowspan="1" colspan="1">Episomal DNA</td><td rowspan="1" colspan="1">Non-integrated circular DNA forms</td></tr><tr><td rowspan="1" colspan="1">Unspliced RNA</td><td rowspan="1" colspan="1">Unprocessed RNA transcripts of HIV (full length HIV)</td></tr><tr><td rowspan="1" colspan="1">Multiple spliced RNA</td><td rowspan="1" colspan="1">Processed RNA HIV transcripts</td></tr><tr><td colspan="2" rowspan="1"><bold><italic>Non PCR-based methods</italic></bold></td></tr><tr><td rowspan="1" colspan="1">Viral outgrowth</td><td rowspan="1" colspan="1">The replication competent fraction of HIV viruses</td></tr></tbody></table></table-wrap><fig id="jve-2-162-g003" orientation="portrait" position="float"><label>Figure 3.</label><caption><p id="jve1-para-0017">Overview of ddPCR HIV assays across all reports. Table view (a) and pie chart (b) of reported use of the assay across all reports based on Table <xref ref-type="table" rid="jve1-tbl-0003">3</xref></p></caption><graphic xlink:href="jve-2-162-g003"></graphic></fig><p id="jve1-para-0018">In the first part of this review, we have focused on reports that have compared ddPCR and qPCR platforms for different types of HIV assays. In the second part, we have described further the applications of ddPCR in HIV research according to the type of HIV assay.</p></sec><sec id="jve1-sec-0007"><title id="jve1-title-0009">Comparison of ddPCR- and qPCR-based assays for HIV quantification</title><p id="jve1-para-0019">Since 2012, five reports have assessed and used ddPCR as a novel quantitative tool in HIV diagnostics and compared it to already-validated qPCR platforms. In this review, we have evaluated both platforms across these reports regarding their technical aspects (Table <xref ref-type="table" rid="jve1-tbl-0003">3</xref>).</p><table-wrap id="jve1-tbl-0003" orientation="portrait" position="float"><label>Table 3.</label><caption><p id="jve1-para-0020">Comparison of ddPCR and qPCR in HIV quantification assays. Colour scale indicates whether qPCR (blue) or ddPCR (green) was superior on a certain technical aspect or when both platforms showed equal performance (orange). –: no data available</p></caption><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col></colgroup><tbody><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">Report</td><td rowspan="1" colspan="1">Virus</td><td rowspan="1" colspan="1">Assay</td><td rowspan="1" colspan="1">Accuracy and bias</td><td rowspan="1" colspan="1">Precision</td><td rowspan="1" colspan="1">Sensitivity</td><td rowspan="1" colspan="1">Limit of detection</td><td rowspan="1" colspan="1">Reproducibility</td></tr><tr><td rowspan="2" colspan="1">1</td><td rowspan="1" colspan="1">Henrich <italic>et al.</italic><xref rid="jve1-bib-0014" ref-type="bibr">[14]</xref></td><td rowspan="1" colspan="1">HIV-1</td><td rowspan="1" colspan="1">Total HIV DNA</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">equal</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">2-LTR circles</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">equal</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td></tr><tr><td rowspan="2" colspan="1">2</td><td rowspan="1" colspan="1">Strain <italic>et al.</italic><xref rid="jve1-bib-0016" ref-type="bibr">[16]</xref></td><td rowspan="1" colspan="1">HIV-1</td><td rowspan="1" colspan="1">Total HIV DNA</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">2-LTR circles</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr</td></tr><tr><td rowspan="2" colspan="1">3</td><td rowspan="1" colspan="1">Kiselinova <italic>et al.</italic><xref rid="jve1-bib-0019" ref-type="bibr">[19]</xref></td><td rowspan="1" colspan="1">HIV-1</td><td rowspan="1" colspan="1">usRNA</td><td rowspan="1" colspan="1">qpcr</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">qpcr</td><td rowspan="1" colspan="1">equal</td><td rowspan="1" colspan="1">–</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">msRNA</td><td rowspan="1" colspan="1">qpcr</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">qpcr</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">–</td></tr><tr><td rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">Bosman <italic>et al.</italic><xref rid="jve1-bib-0028" ref-type="bibr">[28]</xref></td><td rowspan="1" colspan="1">HIV-1</td><td rowspan="1" colspan="1">Total HIV DNA</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">equal</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td></tr><tr><td rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">Ruelle <italic>et al.</italic><xref rid="jve1-bib-0021" ref-type="bibr">[21]</xref></td><td rowspan="1" colspan="1">HIV-2</td><td rowspan="1" colspan="1">RNA (viral load)</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">–</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr</td><td rowspan="1" colspan="1">ddpcr
<break></break></td></tr></tbody></table></table-wrap><sec id="jve1-sec-0008"><title id="jve1-title-0010">Assays for HIV DNA quantification</title><p id="jve1-para-0021">Henrich <italic>et al</italic>. performed the first comparison between ddPCR and qPCR for the quantification of total HIV DNA and 2-LTR circles <xref rid="jve1-bib-0014" ref-type="bibr">[14]</xref>. This was conducted using serial dilutions of DNA standards on samples from HIV-infected patients (<italic>n</italic>=7) who were on or off antiretroviral therapy (ART). The results by qPCR and ddPCR quantification correlated well across all conditions and showed a similar type of detection sensitivity. However, differences in the absolute number of HIV DNA and 2-LTR circles were observed, with consistently higher numbers measured by qPCR. The authors suggested some potential explanations for this observation, including the effect of indirect quantification by standard curve for qPCR (overestimation), or decreased PCR efficiencies for ddPCR caused by incomplete DNA restriction or overloading of the droplets with target DNA copies (underestimation) <xref rid="jve1-bib-0014" ref-type="bibr">[14]</xref>. Indeed, the latter was observed when loading more than 75,000 DNA copies per droplet, showing the need for samples to be diluted beyond this point. Finally, the authors have commented on the need for automated and reproducible threshold determination methods in order to increase the reproducibility of ddPCR quantification.</p><p id="jve1-para-0022">A second comparison between ddPCR and qPCR for the quantification of total HIV DNA and 2-LTR circles was made by Strain <italic>et al</italic>., which involved an analysis of over 300 clinical samples <xref rid="jve1-bib-0016" ref-type="bibr">[16]</xref>. Here, ddPCR has shown an improved precision for total HIV DNA quantification and better accuracy for the quantification of 2-LTR circles. Furthermore, the authors reported that ddPCR offers an extra benefit for the quantification of micro-organisms with high sequence variability, such as HIV, because of an increased robustness to the mismatch of primers/probes with the target sequence. Indeed, in their experimental setup, mismatches were better tolerated by ddPCR than by qPCR, resulting in more accurate quantification results. Lastly, Strain <italic>et al</italic>. observed the existence of low numbers of false positive droplets in the negative template control (NTC) and presented a new data-driven threshold determination method to address the suboptimal threshold calculations made by ddPCR manufacturers’ software (Quantasoft, Bio-Rad).</p><p id="jve1-para-0023">A third and final comparison for a total HIV DNA assay was conducted by Bosman <italic>et al</italic>. <xref rid="jve1-bib-0028" ref-type="bibr">[28]</xref> and included two ddPCR platforms (QX100, Bio-Rad and Raindrop, Raindance) and a semi-nested qPCR on serial HIV DNA dilutions and samples from ART-suppressed patients <xref rid="jve1-bib-0028" ref-type="bibr">[28]</xref>. Semi-nested qPCRs are specifically designed to reach higher sensitivity and accuracy as compared to the standard qPCR. The QX100 ddPCR showed equal sensitivity with the semi-nested qPCR but higher precision and efficiency, corroborating the findings by Strain <italic>et al</italic>. <xref rid="jve1-bib-0016" ref-type="bibr">[16]</xref>. However, low numbers of false positive droplets in the NTCs were observed in the ddPCR. These droplets displayed high fluorescence, making them indistinguishable from the positive droplets in positive control samples. The semi-nested qPCR did not result in false positive samples. Hence, it was concluded that ddPCR outperforms qPCR for accurate quantification of low levels of HIV DNA, but that a semi-nested qPCR platform may be preferred if the focus is on discriminating between the presence or absence of HIV DNA.</p></sec><sec id="jve1-sec-0009"><title id="jve1-title-0011">Assays for HIV RNA quantification</title><p id="jve1-para-0024">Two reports have discussed the comparison of qPCR and ddPCR for HIV RNA assays, one for HIV-1 and one for HIV-2 <xref rid="jve1-bib-0019" ref-type="bibr">[19,21]</xref>. Kiselinova <italic>et al</italic>. compared a semi-nested qPCR and ddPCR for the quantification of cell-associated HIV-1 RNA (unspliced and multiple spliced) <xref rid="jve1-bib-0019" ref-type="bibr">[19]</xref>. This study highlighted that, in contrast to DNA quantification, RNA quantification by dPCR still requires a calibrator to correct for reverse transcription efficiency when transforming the template RNA to PCR-compatible cDNA. Furthermore, in this report, semi-nested qPCR showed better accuracy and sensitivity on a synthetic RNA standard, especially in the lower range of quantification. In contrast, ddPCR detected multiple spliced HIV-1 RNA in a larger portion of patients, resulting in higher sensitivity for that assay. However, a low number of false positive droplets in the NTC were observed in the ddPCR readouts (as described elsewhere <xref rid="jve1-bib-0016" ref-type="bibr">[16,28]</xref>). Interestingly, Kiselinova <italic>et al</italic>. <xref rid="jve1-bib-0019" ref-type="bibr">[19]</xref> have also reported false positive droplets in NTCs in assays where either the forward or reverse primer was lacking, but with the other primer and probe in the reaction. This indicates that false positive droplets do not always arise from low levels of contamination, but may be artefacts from the specific type of assay and platform.</p><p id="jve1-para-0025">Concerning HIV-2 quantification, Ruelle <italic>et al</italic>. <xref rid="jve1-bib-0021" ref-type="bibr">[21]</xref> presented an optimised ddPCR assay for plasma-free HIV-2 RNA (plasma viral load) measurement and found that ddPCR represented a more reproducible and sensitive assay as compared with qPCR. However, these authors have confirmed that false positive droplets in NTC remain an issue and underlined the need for an accurate threshold determination method before a transfer of ddPCR platforms into clinical testing.</p></sec></sec><sec id="jve1-sec-0010"><title id="jve1-title-0012">ddPCR applications in HIV reservoir quantification studies</title><p id="jve1-para-0026">Droplet digital PCR has been increasingly used in different research settings to address specific HIV-related topics. Across all reports, ddPCR was most often used for the quantification of HIV DNA (total HIV DNA and 2-LTR) in the context of the latent HIV reservoir (Figure <xref ref-type="fig" rid="jve-2-162-g003">3</xref>). In addition, different body compartments were targeted for absolute ddPCR HIV quantification, such as blood, rectal tissue and cerebrospinal fluid (CSF).</p><sec id="jve1-sec-0011"><title id="jve1-title-0013">Total HIV DNA</title><p id="jve1-para-0027">de Oliveira <italic>et al</italic>. (2015) measured cell-associated HIV DNA levels by ddPCR in the CSF and paired peripheral blood mononuclear cell (PBMC) samples from 28 patients, 19 of whom were ART suppressed with an undetectable plasma HIV RNA viral load (<50 HIV copies/mL) <xref rid="jve1-bib-0030" ref-type="bibr">[30]</xref>. They found that HIV DNA levels in CSF were comparable between ART-suppressed and non-suppressed patients, but lower in PBMCs of suppressed patients. The authors concluded that the CSF showed lower ART penetration and can harbour ongoing viral replication with replenishment of the viral reservoir.</p><p id="jve1-para-0028">King <italic>et al</italic>. performed ddPCR for HIV DNA detection in infants breast fed by HIV-infected mothers who were receiving post-natal antiretroviral prophylaxis <xref rid="jve1-bib-0029" ref-type="bibr">[29]</xref>. Here, ddPCR HIV DNA quantification showed delayed detection of the virus with prophylaxis, resulting in missed diagnosis in infants when HIV testing was performed within 6 weeks after breastfeeding cessation.</p><p id="jve1-para-0029">In addition, a study performed by Yukl <italic>et al</italic>. <xref rid="jve1-bib-0018" ref-type="bibr">[18]</xref> with blood and tissue samples from the so-called Berlin patient who was reported cured from HIV after receiving a bone marrow transplant from a homozygous CCR5Δ32 donor <xref rid="jve1-bib-0032" ref-type="bibr">[32]</xref>. Here, ddPCR was used to characterise total HIV DNA in PBMCs in order to assess the impact of potential curative interventions by bone marrow transplantation <xref rid="jve1-bib-0018" ref-type="bibr">[18]</xref>. No HIV DNA was detected in PBMCs but low levels of HIV DNA were detected by qPCR in rectum samples. The authors state that these low levels might represent a false positive result as no HIV sequences could be retrieved from rectum samples and HIV levels were near the limit of detection of the qPCR assay.</p></sec><sec id="jve1-sec-0012"><title id="jve1-title-0014">2-LTR circles</title><p id="jve1-para-0030">Hatano <italic>et al</italic>. monitored 2-LTR circles by means of ddPCR in a randomised control study in HIV-suppressed patients receiving either raltegravir intensification (<italic>n</italic>=15) or a placebo (<italic>n</italic>=16) <xref rid="jve1-bib-0023" ref-type="bibr">[23]</xref>. They found an increase of 2-LTR circles over time in the group receiving raltegravir. The authors state that this might indicate a low level of ongoing replication that can be blocked by raltegravir intensification in ART-suppressed patients.</p><p id="jve1-para-0031">Another study that has looked at ART-suppressed patients (<italic>n</italic>=50) was conducted by Ruggiero <italic>et al</italic>. <xref rid="jve1-bib-0024" ref-type="bibr">[24]</xref>. Here, ddPCR was used in the determination of 2-LTR circles in addition to a range of other PCR-based HIV quantification assays and flow cytometry for immunological markers (CD4 plus one of CD26/CD38/CD69, CD8 plus either one of CD38/HLA-DR/DP/DQ). No correlation was found between 2-LTR circles with the HIV reservoir or the immunological markers studied.</p></sec><sec id="jve1-sec-0013"><title id="jve1-title-0015">Total HIV DNA and 2-LTR circles</title><p id="jve1-para-0032">Pallikuth <italic>et al</italic>. looked into HIV permissiveness and persistence in a subset of peripheral blood central memory T cells, namely peripheral T follicular helper cells (Tfh) <xref rid="jve1-bib-0025" ref-type="bibr">[25]</xref>. Total HIV DNA and 2-LTR circles were measured in chronically infected patients before and after 48 weeks of raltegravir-based ART. Total HIV levels were found to remain stable but those of 2-LTR circles decreased over 48 weeks of treatment in Tfh cells.</p><p id="jve1-para-0033">Eriksson <italic>et al</italic>. used ddPCR to monitor total HIV DNA and 2-LTR circles and compared them using a non-PCR-based method that detects replication-competent HIV (i.e. the viral outgrowth assay) <xref rid="jve1-bib-0015" ref-type="bibr">[15]</xref>. They found a discrepancy between the two methods, in so far as PCR-based methods recorded higher levels of HIV than those of replication-competent virus detected by the viral outgrowth assays (VOA). The authors state that this might be due to the substantial amount of replication-deficient HIV sequences found in HIV-infected patients that are picked up by PCR-based assays but not by the VOA <xref rid="jve1-bib-0015" ref-type="bibr">[15]</xref>. In addition, more recent results indicate that the VOA underestimates the amount of replication-competent HIV DNA <xref rid="jve1-bib-0033" ref-type="bibr">[33]</xref>.</p></sec><sec id="jve1-sec-0014"><title id="jve1-title-0016">Total HIV DNA and cell-associated HIV RNA</title><p id="jve1-para-0034">Three studies have assessed ddPCR for total HIV DNA and cell-associated HIV RNA quantification in various patient cohorts. A first study by Kiselinova <italic>et al</italic>. used ddPCR in HIV RNA and DNA measurements for the comparison of different ART regimens: a standard ART backbone (two nucleoside reverse transcriptase inhibitors) combined with nevirapine or a protease inhibitor <xref rid="jve1-bib-0022" ref-type="bibr">[22,23]</xref>. No difference in HIV RNA and DNA was found between the two treatment regimens. A second study by Malatinkova <italic>et al</italic>. determined HIV DNA and RNA levels with ddPCR in two body compartments (blood and rectal tissue) in different patient cohorts: early treated seroconverters, chronically treated patients, ART-naïve seroconverters and long-term non-progressors (LTNP) <xref rid="jve1-bib-0024" ref-type="bibr">[24]</xref>. This study confirmed that early treatment is associated with a small viral reservoir and low levels of RNA transcription as compared to later treatment initiation. However, total HIV DNA levels were still higher than those of LTNPs <xref rid="jve1-bib-0024" ref-type="bibr">[24]</xref>. In a third study, Kiselinova <italic>et al</italic>. have looked further into the discrepancy between the quantification results of the VOA and HIV DNA- and RNA PCR-based methods in samples of patients on ART <xref rid="jve1-bib-0031" ref-type="bibr">[31]</xref>. Here, total HIV DNA levels were found to be higher than the replication-competent levels measured by the VOA, corroborating earlier findings by Eriksson <italic>et al</italic>. <xref rid="jve1-bib-0015" ref-type="bibr">[15]</xref>. Additionally, the pool of total HIV DNA correlated well with the replication-competent reservoir measured by the VOA. Hence, the authors concluded that the amount of latent replication-competent HIV in patients receiving treatment can be predicted by measuring total HIV DNA levels using PCR-based methods <xref rid="jve1-bib-0031" ref-type="bibr">[31]</xref>. Therefore, these methods can still be a useful tool in HIV eradication studies.</p><p id="jve1-para-0035">A last example of the use of ddPCR for the quantification of cell-associated HIV-1 RNA and DNA is in HIV cure trials aimed at eliminating the HIV latent reservoir. In this context, several groups have reported the effect of various latency-reversing agents (e.g. panobinostat, romidepsin) on stimulating the latent reservoir <xref rid="jve1-bib-0020" ref-type="bibr">[20,26]</xref>.</p></sec></sec></sec><sec id="jve1-sec-0015"><title id="jve1-title-0017">Discussion</title><p id="jve1-para-0036">There has been renewed interest in the concept of digital PCR since the introduction of new commercial (droplet) digital PCR platforms <xref rid="jve1-bib-0006" ref-type="bibr">[6,9]</xref>. The dPCR platforms have promised to be less labour-intensive with lower turnaround times, which should be attractive to many HIV research groups. Hence, comparison with established qPCR platforms for nucleic acid quantification is required.</p><p id="jve1-para-0037">The findings of this review confirm reports on ddPCR and qPCR comparisons within the field of cancer diagnostics (prostate cancer) and quantification of other viruses (e.g. cytomegalovirus), suggesting that ddPCR offers greater precision, improved reproducibility but with similar sensitivity to qPCR <xref rid="jve1-bib-0001" ref-type="bibr">[1–3]</xref>. Furthermore, ddPCR benefits from direct quantification without the need for standard curves and offers a platform that displays higher robustness to primer and probe mismatches with the target sequence, making this a desired platform for HIV quantification as sequence heterogeneity is often observed. However, it must be noted that the difference between ddPCR and the more sensitive semi-nested qPCR is less clear <xref rid="jve1-bib-0019" ref-type="bibr">[19,28]</xref>. None the less, the transition from qPCR to ddPCR could simplify and improve routine clinical/virological testing. However, before ddPCR can be fully accepted as an improved alternative to qPCR, two remaining issues have been reported that are discussed below.</p><p id="jve1-para-0038">The first involves ddPCR sensitivity and the presence of false positive droplets. Multiple reports have stated the existence of highly fluorescent droplets in negative template controls containing template-free genomic DNA, referred to as ‘false positive droplets’ <xref rid="jve1-bib-0016" ref-type="bibr">[16,19,28]</xref>. This phenomenon seems inherent to ddPCR platforms (i.e. QX100 and QX200 Bio-Rad) and lowers the sensitivity of ddPCR quantification. This issue is especially problematic in the context of a low level of viral detection, as is the case for HIV quantification in patients receiving optimal ART with suppressed plasma viral load <xref rid="jve1-bib-0014" ref-type="bibr">[14,16]</xref>. Therefore, extra quality-assurance steps are mandatory and crucial in order to identify and exclude false positive droplets and to achieve a higher sensitivity. In this context, new dPCR technologies that implement quality control for identifying false positive droplets could offer a (hardware) solution for this problem.</p><p id="jve1-para-0039">The second issue involves the standardised threshold determination. Ruelle <italic>et al</italic>. commented on the need for a standardised automated threshold setting, as the software methods provided by the current ddPCR platform manufacturers are not disclosed and often result in suboptimal quantification <xref rid="jve1-bib-0021" ref-type="bibr">[21]</xref>. In this context, three alternative threshold determination methods are described to further improve reproducible data analysis: clustering methods <xref rid="jve1-bib-0016" ref-type="bibr">[16,34]</xref>, global manual threshold <xref rid="jve1-bib-0035" ref-type="bibr">[35]</xref> and ddpcRquant <xref rid="jve1-bib-0036" ref-type="bibr">[36]</xref>.</p><p id="jve1-para-0040">Strain <italic>et al</italic>. were first to suggest a clustering-based method to identify positive and negative droplet populations by calculating two thresholds to delineate these two populations. Droplets that fall outside these intervals, referred to as ‘rain’, are discarded and excluded from further concentration calculations <xref rid="jve1-bib-0016" ref-type="bibr">[16]</xref>. Jones <italic>et al</italic>. have discussed a similar approach, ‘definetherain’, which is based on k-nearest neighbour clustering to discriminate negative from positive droplet populations <xref rid="jve1-bib-0034" ref-type="bibr">[34]</xref>.</p><p id="jve1-para-0041">However, our group has shown that droplets considered as rain can contain genuine HIV sequences with mismatches in the primer or probe binding region <xref rid="jve1-bib-0036" ref-type="bibr">[36]</xref>. Hence, the elimination of droplets can bias quantitative outcomes, especially in the context of some viral or bacterial genomes where a high frequency in sequence variation is observed <xref rid="jve1-bib-0016" ref-type="bibr">[16]</xref>. The global manual threshold method offers a single threshold determination that is calculated as six times the standard deviation of the negative droplet population and, in contrast to definetherain, it includes droplets with intermediate fluorescence (rain) <xref rid="jve1-bib-0035" ref-type="bibr">[35]</xref>. Both the definetherain and global manual threshold data analysis methods assume a normal distribution of the droplet fluorescence. However, this assumption seldom holds true and can affect the correct allocation of thresholds or clusters <xref rid="jve1-bib-0036" ref-type="bibr">[36]</xref>. Therefore, we have recently described an alternative statistical framework, ddpcRquant, to allocate thresholds based on an extreme value theory, which is independent of the underlying distribution of the total negative droplets. The ddpcRquant algorithm also accounts for shifts in baseline fluorescence between samples that can alter the correct droplet allocation (code and web tool interface available at
<uri xlink:href="http://www.ddpcrquant.ugent.be">www.ddpcrquant.ugent.be</uri>) <xref rid="jve1-bib-0036" ref-type="bibr">[36]</xref>.</p></sec><sec id="jve1-sec-0016"><title id="jve1-title-0018">Conclusion and future perspectives</title><p id="jve1-para-0042">There is growing interest in improving the sensitivity/detection limit of qPCR. In this context, dPCR platforms could represent an improved solution. However, the currently available platforms still struggle to fulfil these promises and the needs of the different end-users across multiple research domains. For the application in viral detection, ddPCR holds an inherent advantage over qPCR because of higher robustness to mismatches between the template and primers/probe. On the other hand, the reports of false positive droplets in NTCs remain a hurdle when attempting to reach higher sensitivity and, although new threshold determination methods offer a more reliable means of quantification, they are not applicable to all ddPCR experimental setups. These issues will need to be further addressed with costs to be further reduced in order for ddPCR to make its way into the clinically accredited setting.</p></sec></body><back><ack id="jve1-ack-0001"><title id="jve1-title-0019">Acknowledgements</title></ack><sec id="jve1-sec-0017"><title id="jve1-title-0020"></title><sec id="jve1-sec-0018"><title id="jve1-title-0021">Declaration of interest</title><p id="jve1-para-0043">The authors have no competing interests to declare.</p></sec></sec><sec id="jve1-sec-0019"><title id="jve1-title-0022"></title><sec id="jve1-sec-0020"><title id="jve1-title-0023">Funding</title><p id="jve1-para-0044">The authors would like to acknowledge the support of following research grants: Amfar (Group funding, grant 108314-51-RGRL), HIVERA/SBO IWT (Group funding, grant 130442), FWO (Linos Vandekerckhove, grant 1.8.020.09.N.00), FWO (Ward De Spiegelaere, grant 12G9716N), BOF (Maja Kiselinova, grant 01N02712), King Baudouin Foundation (Group funding, grant 2010-R20640-003), unrestricted grant of Bristol-Myers Squibb Belgium (Group funding).</p></sec></sec><ref-list id="jve1-ref-0001"><title>References</title><ref id="jve1-bib-0001"><label>1.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Huggett</surname><given-names>JF</given-names></name>,
<name name-style="western"><surname>Cowen</surname><given-names>S</given-names></name>,
<name name-style="western"><surname>Foy</surname><given-names>CA</given-names></name></person-group><article-title>Considerations for digital PCR as an accurate molecular diagnostic tool</article-title>.
<source>Clin Chem</source><year>2015</year>;
<volume>61</volume>:
<fpage>79</fpage>–
<lpage>88</lpage>.<pub-id pub-id-type="pmid">25338683</pub-id></mixed-citation></ref><ref id="jve1-bib-0002"><label>2.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hindson</surname><given-names>CM</given-names></name>,
<name name-style="western"><surname>Chevillet</surname><given-names>JR</given-names></name>,
<name name-style="western"><surname>Briggs</surname><given-names>HA</given-names></name><etal>et al.</etal></person-group><article-title>Absolute quantification by droplet digital PCR versus analog real-time PCR</article-title>.
<source>Nat Methods</source><year>2013</year>;
<volume>10</volume>:
<fpage>1003</fpage>–
<lpage>1005</lpage>.<pub-id pub-id-type="pmid">23995387</pub-id></mixed-citation></ref><ref id="jve1-bib-0003"><label>3.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Sedlak</surname><given-names>RH</given-names></name>,
<name name-style="western"><surname>Jerome</surname><given-names>KR</given-names></name></person-group><article-title>Viral diagnostics in the era of digital polymerase chain reaction</article-title>.
<source>Diagn Microbiol Infect Dis</source><year>2013</year>;
<volume>75</volume>:
<fpage>1</fpage>–
<lpage>4</lpage>.<pub-id pub-id-type="pmid">23182074</pub-id></mixed-citation></ref><ref id="jve1-bib-0004"><label>4.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Simmonds</surname><given-names>P</given-names></name>,
<name name-style="western"><surname>Balfe</surname><given-names>P</given-names></name>,
<name name-style="western"><surname>Peutherer</surname><given-names>JF</given-names></name><etal>et al.</etal></person-group><article-title>Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers</article-title>.
<source>J Virol</source><year>1990</year>;
<volume>64</volume>:
<fpage>864</fpage>–
<lpage>872</lpage>.<pub-id pub-id-type="pmid">2296085</pub-id></mixed-citation></ref><ref id="jve1-bib-0005"><label>5.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hindson</surname><given-names>BJ</given-names></name>,
<name name-style="western"><surname>Ness</surname><given-names>KD</given-names></name>,
<name name-style="western"><surname>Masquelier</surname><given-names>DA</given-names></name><etal>et al.</etal></person-group><article-title>High-throughput droplet digital PCR system for absolute quantitation of DNA copy number</article-title>.
<source>Anal Chem</source><year>2011</year>;
<volume>83</volume>:
<fpage>8604</fpage>–
<lpage>8610</lpage>.<pub-id pub-id-type="pmid">22035192</pub-id></mixed-citation></ref><ref id="jve1-bib-0006"><label>6.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Pinheiro</surname><given-names>LB</given-names></name>,
<name name-style="western"><surname>Coleman</surname><given-names>VA</given-names></name>,
<name name-style="western"><surname>Hindson</surname><given-names>CM</given-names></name><etal>et al.</etal></person-group><article-title>Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification</article-title>.
<source>Anal Chem</source><year>2012</year>;
<volume>84</volume>:
<fpage>1003</fpage>–
<lpage>1011</lpage>.<pub-id pub-id-type="pmid">22122760</pub-id></mixed-citation></ref><ref id="jve1-bib-0007"><label>7.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Vogelstein</surname><given-names>B</given-names></name>,
<name name-style="western"><surname>Kinzler</surname><given-names>KW</given-names></name></person-group><article-title>Digital PCR</article-title>.
<source>Proc Natl Acad Sci U S A</source><year>1999</year>;
<volume>96</volume>:
<fpage>9236</fpage>–
<lpage>9241</lpage>.<pub-id pub-id-type="pmid">10430926</pub-id></mixed-citation></ref><ref id="jve1-bib-0008"><label>8.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Morley</surname><given-names>AA</given-names></name></person-group><article-title>Digital PCR: A brief history</article-title>.
<source>Biomol Detect Quantif</source><year>2014</year>;
<volume>1</volume>:
<fpage>1</fpage>–
<lpage>2</lpage>.</mixed-citation></ref><ref id="jve1-bib-0009"><label>9.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Taly</surname><given-names>V</given-names></name>,
<name name-style="western"><surname>Pekin</surname><given-names>D</given-names></name>,
<name name-style="western"><surname>El Abed</surname><given-names>A</given-names></name>,
<name name-style="western"><surname>Laurent-Puig</surname><given-names>P</given-names></name></person-group><article-title>Detecting biomarkers with microdroplet technology</article-title>.
<source>Trends Mol Med</source><year>2012</year>;
<volume>18</volume>:
<fpage>405</fpage>–
<lpage>416</lpage>.<pub-id pub-id-type="pmid">22727782</pub-id></mixed-citation></ref><ref id="jve1-bib-0010"><label>10.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Strain</surname><given-names>MC</given-names></name>,
<name name-style="western"><surname>Richman</surname><given-names>DD</given-names></name></person-group><article-title>New assays for monitoring residual HIV burden in effectively treated individuals</article-title>.
<source>Curr Opin HIV AIDS</source><year>2013</year>;
<volume>8</volume>:
<fpage>106</fpage>–
<lpage>110</lpage>.<pub-id pub-id-type="pmid">23314907</pub-id></mixed-citation></ref><ref id="jve1-bib-0011"><label>11.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Theodorsson</surname><given-names>E</given-names></name>,
<name name-style="western"><surname>Magnusson</surname><given-names>B</given-names></name>,
<name name-style="western"><surname>Leito</surname><given-names>I</given-names></name></person-group><article-title>Bias in clinical chemistry</article-title>.
<source>Bioanalysis</source><year>2014</year>;
<volume>6</volume>:
<fpage>2855</fpage>–
<lpage>2875</lpage>.<pub-id pub-id-type="pmid">25486232</pub-id></mixed-citation></ref><ref id="jve1-bib-0012"><label>12.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Westgard</surname><given-names>JO</given-names></name></person-group><article-title>Useful measures and models for analytical quality management in medical laboratories</article-title>.
<source>Clin Chem Lab Med</source><year>2016</year>;
<volume>54</volume>:
<fpage>223</fpage>–
<lpage>233</lpage>.<pub-id pub-id-type="pmid">26426893</pub-id></mixed-citation></ref><ref id="jve1-bib-0013"><label>13.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Armbruster</surname><given-names>DA</given-names></name>,
<name name-style="western"><surname>Pry</surname><given-names>T</given-names></name></person-group><article-title>Limit of blank, limit of detection and limit of quantitation</article-title>.
<source>Clin Biochem Rev</source><year>2008</year>;
<volume>29</volume><issue>Suppl 1</issue>:
<fpage>S49</fpage>–
<lpage>52</lpage>.<pub-id pub-id-type="pmid">18852857</pub-id></mixed-citation></ref><ref id="jve1-bib-0014"><label>14.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Henrich</surname><given-names>TJ</given-names></name>,
<name name-style="western"><surname>Gallien</surname><given-names>S</given-names></name>,
<name name-style="western"><surname>Li</surname><given-names>JZ</given-names></name><etal>et al.</etal></person-group><article-title>Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR</article-title>.
<source>J Virol Methods</source><year>2012</year>;
<volume>186</volume>:
<fpage>68</fpage>–
<lpage>72</lpage>.<pub-id pub-id-type="pmid">22974526</pub-id></mixed-citation></ref><ref id="jve1-bib-0015"><label>15.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Eriksson</surname><given-names>S</given-names></name>,
<name name-style="western"><surname>Graf</surname><given-names>EH</given-names></name>,
<name name-style="western"><surname>Dahl</surname><given-names>V</given-names></name><etal>et al.</etal></person-group><article-title>Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies</article-title>.
<source>PLoS Pathog</source><year>2013</year>;
<volume>9</volume>:
<fpage>e1003174</fpage>.<pub-id pub-id-type="pmid">23459007</pub-id></mixed-citation></ref><ref id="jve1-bib-0016"><label>16.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Strain</surname><given-names>MC</given-names></name>,
<name name-style="western"><surname>Lada</surname><given-names>SM</given-names></name>,
<name name-style="western"><surname>Luong</surname><given-names>T</given-names></name><etal>et al.</etal></person-group><article-title>Highly precise measurement of HIV DNA by droplet digital PCR</article-title>.
<source>PLoS One</source><year>2013</year>;
<volume>8</volume>:
<fpage>e55943</fpage>.<pub-id pub-id-type="pmid">23573183</pub-id></mixed-citation></ref><ref id="jve1-bib-0017"><label>17.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hatano</surname><given-names>H</given-names></name>,
<name name-style="western"><surname>Strain</surname><given-names>MC</given-names></name>,
<name name-style="western"><surname>Scherzer</surname><given-names>R</given-names></name><etal>et al.</etal></person-group><article-title>Increase in 2-long terminal repeat circles and decrease in D-dimer after raltegravir intensification in patients with treated HIV infection: a randomized, placebo-controlled trial</article-title>.
<source>J Infect Dis</source><year>2013</year>;
<volume>208</volume>:
<fpage>1436</fpage>–
<lpage>1442</lpage>.<pub-id pub-id-type="pmid">23975885</pub-id></mixed-citation></ref><ref id="jve1-bib-0018"><label>18.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Yukl</surname><given-names>SA</given-names></name>,
<name name-style="western"><surname>Boritz</surname><given-names>E</given-names></name>,
<name name-style="western"><surname>Busch</surname><given-names>M</given-names></name><etal>et al.</etal></person-group><article-title>Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient</article-title>.
<source>PLoS Pathog</source><year>2013</year>;
<volume>9</volume>:
<fpage>e1003347</fpage>.<pub-id pub-id-type="pmid">23671416</pub-id></mixed-citation></ref><ref id="jve1-bib-0019"><label>19.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Kiselinova</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Pasternak</surname><given-names>AO</given-names></name>,
<name name-style="western"><surname>De Spiegelaere</surname><given-names>W</given-names></name><etal>et al.</etal></person-group><article-title>Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA</article-title>.
<source>PLoS One</source><year>2014</year>;
<volume>9</volume>:
<fpage>e85999</fpage>.<pub-id pub-id-type="pmid">24465831</pub-id></mixed-citation></ref><ref id="jve1-bib-0020"><label>20.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Rasmussen</surname><given-names>TA</given-names></name>,
<name name-style="western"><surname>Tolstrup</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Brinkmann</surname><given-names>CR</given-names></name><etal>et al.</etal></person-group><article-title>Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial</article-title>.
<source>Lancet HIV</source><year>2014</year>;
<volume>1</volume>:
<fpage>e13</fpage>–
<lpage>21</lpage>.<pub-id pub-id-type="pmid">26423811</pub-id></mixed-citation></ref><ref id="jve1-bib-0021"><label>21.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Ruelle</surname><given-names>J</given-names></name>,
<name name-style="western"><surname>Yfantis</surname><given-names>V</given-names></name>,
<name name-style="western"><surname>Duquenne</surname><given-names>A</given-names></name>,
<name name-style="western"><surname>Goubau</surname><given-names>P</given-names></name></person-group><article-title>Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification</article-title>.
<source>J Int AIDS Soc</source><year>2014</year>;
<volume>17</volume>:
<fpage>19675</fpage>.<pub-id pub-id-type="pmid">25397425</pub-id></mixed-citation></ref><ref id="jve1-bib-0022"><label>22.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Kiselinova</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Anna</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Malatinkova</surname><given-names>E</given-names></name><etal>et al.</etal></person-group><article-title>The impact of nevirapine- versus protease inhibitor-based regimens on virological markers of HIV-1 persistence during seemingly suppressive ART</article-title>.
<source>J Int AIDS Soc</source><year>2014</year>;
<volume>17</volume>:
<fpage>19823</fpage>.<pub-id pub-id-type="pmid">25397567</pub-id></mixed-citation></ref><ref id="jve1-bib-0023"><label>23.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Kiselinova</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Geretti</surname><given-names>AM</given-names></name>,
<name name-style="western"><surname>Malatinkova</surname><given-names>E</given-names></name><etal>et al.</etal></person-group><article-title>HIV-1 RNA and HIV-1 DNA persistence during suppressive ART with PI-based or nevirapine-based regimens</article-title>.
<source>J Antimicrob Chemother</source><year>2015</year>;
<volume>70</volume>:
<fpage>3311</fpage>–
<lpage>3316</lpage>.<pub-id pub-id-type="pmid">26324076</pub-id></mixed-citation></ref><ref id="jve1-bib-0024"><label>24.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Malatinkova</surname><given-names>E</given-names></name>,
<name name-style="western"><surname>De Spiegelaere</surname><given-names>W</given-names></name>,
<name name-style="western"><surname>Bonczkowski</surname><given-names>P</given-names></name><etal>et al.</etal></person-group><article-title>Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs</article-title>.
<source>Elife</source><year>2015</year>;
<volume>4</volume>:
<fpage>e09115</fpage>.<pub-id pub-id-type="pmid">26439007</pub-id></mixed-citation></ref><ref id="jve1-bib-0025"><label>25.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Pallikkuth</surname><given-names>S</given-names></name>,
<name name-style="western"><surname>Sharkey</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Babic</surname><given-names>DZ</given-names></name><etal>et al.</etal></person-group><article-title>Peripheral T follicular helper cells are the major HIV reservoir within central memory CD4 T cells in peripheral blood from chronically HIV-infected individuals on combination antiretroviral therapy</article-title>.
<source>J Virol</source><year>2015</year>;
<volume>90</volume>:
<fpage>2718</fpage>–
<lpage>2728</lpage>.<pub-id pub-id-type="pmid">26676775</pub-id></mixed-citation></ref><ref id="jve1-bib-0026"><label>26.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Sogaard</surname><given-names>OS</given-names></name>,
<name name-style="western"><surname>Graversen</surname><given-names>ME</given-names></name>,
<name name-style="western"><surname>Leth</surname><given-names>S</given-names></name><etal>et al.</etal></person-group><article-title>The depsipeptide romidepsin reverses HIV-1 latency <italic>in vivo</italic></article-title>.
<source>PLoS Pathog</source><year>2015</year>;
<volume>11</volume>:
<fpage>e1005142</fpage>.<pub-id pub-id-type="pmid">26379282</pub-id></mixed-citation></ref><ref id="jve1-bib-0027"><label>27.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Ruggiero</surname><given-names>A</given-names></name>,
<name name-style="western"><surname>De Spiegelaere</surname><given-names>W</given-names></name>,
<name name-style="western"><surname>Cozzi-Lepri</surname><given-names>A</given-names></name><etal>et al.</etal></person-group><article-title>During stably suppressive antiretroviral therapy integrated HIV-1 DNA load in peripheral blood is associated with the frequency of CD8 cells expressing HLA-DR/DP/DQ</article-title>.
<source>EBioMedicine</source><year>2015</year>;
<volume>2</volume>:
<fpage>1153</fpage>–
<lpage>1159</lpage>.<pub-id pub-id-type="pmid">26498496</pub-id></mixed-citation></ref><ref id="jve1-bib-0028"><label>28.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Bosman</surname><given-names>KJ</given-names></name>,
<name name-style="western"><surname>Nijhuis</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Ham</surname><given-names>PM</given-names><prefix>van</prefix></name><etal>et al.</etal></person-group><article-title>Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir</article-title>.
<source>Sci Rep</source><year>2015</year>;
<volume>5</volume>:
<fpage>13811</fpage>.<pub-id pub-id-type="pmid">26350506</pub-id></mixed-citation></ref><ref id="jve1-bib-0029"><label>29.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>King</surname><given-names>CC</given-names></name>,
<name name-style="western"><surname>Kourtis</surname><given-names>AP</given-names></name>,
<name name-style="western"><surname>Persaud</surname><given-names>D</given-names></name><etal>et al.</etal></person-group><article-title>Delayed HIV detection among infants exposed to postnatal antiretroviral prophylaxis during breastfeeding</article-title>.
<source>AIDS</source><year>2015</year>;
<volume>29</volume>:
<fpage>1953</fpage>–
<lpage>1961</lpage>.<pub-id pub-id-type="pmid">26153671</pub-id></mixed-citation></ref><ref id="jve1-bib-0030"><label>30.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Oliveira</surname><given-names>MF</given-names><prefix>de</prefix></name>,
<name name-style="western"><surname>Gianella</surname><given-names>S</given-names></name>,
<name name-style="western"><surname>Letendre</surname><given-names>S</given-names></name><etal>et al.</etal></person-group><article-title>Comparative analysis of cell-associated HIV DNA levels in cerebrospinal Fluid and peripheral blood by droplet digital PCR</article-title>.
<source>PLoS One</source><year>2015</year>;
<volume>10</volume>:
<fpage>e0139510</fpage>.<pub-id pub-id-type="pmid">26431315</pub-id></mixed-citation></ref><ref id="jve1-bib-0031"><label>31.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Kiselinova</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>De Spiegelaere</surname><given-names>W</given-names></name>,
<name name-style="western"><surname>Buzon</surname><given-names>MJ</given-names></name><etal>et al.</etal></person-group><article-title>Integrated and total HIV-1 DNA predict <italic>ex vivo</italic> Viral outgrowth</article-title>.
<source>PLoS Pathog</source><year>2016</year>;
<volume>12</volume>:
<fpage>e1005472</fpage>.<pub-id pub-id-type="pmid">26938995</pub-id></mixed-citation></ref><ref id="jve1-bib-0032"><label>32.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hutter</surname><given-names>G</given-names></name>,
<name name-style="western"><surname>Nowak</surname><given-names>D</given-names></name>,
<name name-style="western"><surname>Mossner</surname><given-names>M</given-names></name><etal>et al.</etal></person-group><article-title>Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation</article-title>.
<source>N Engl J Med</source><year>2009</year>;
<volume>360</volume>:
<fpage>692</fpage>–
<lpage>698</lpage>.<pub-id pub-id-type="pmid">19213682</pub-id></mixed-citation></ref><ref id="jve1-bib-0033"><label>33.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Ho</surname><given-names>YC</given-names></name>,
<name name-style="western"><surname>Shan</surname><given-names>L</given-names></name>,
<name name-style="western"><surname>Hosmane</surname><given-names>NN</given-names></name><etal>et al.</etal></person-group><article-title>Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure</article-title>.
<source>Cell</source><year>2013</year>;
<volume>155</volume>:
<fpage>540</fpage>–
<lpage>551</lpage>.<pub-id pub-id-type="pmid">24243014</pub-id></mixed-citation></ref><ref id="jve1-bib-0034"><label>34.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Jones</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Williams</surname><given-names>J</given-names></name>,
<name name-style="western"><surname>Gartner</surname><given-names>K</given-names></name><etal>et al.</etal></person-group><article-title>Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain</article-title>’.
<source>J Virol Methods</source><year>2014</year>;
<volume>202</volume>:
<fpage>46</fpage>–
<lpage>53</lpage>.<pub-id pub-id-type="pmid">24598230</pub-id></mixed-citation></ref><ref id="jve1-bib-0035"><label>35.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Dreo</surname><given-names>T</given-names></name>,
<name name-style="western"><surname>Pirc</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>Ramsak</surname><given-names>Z</given-names></name><etal>et al.</etal></person-group><article-title>Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot</article-title>.
<source>Anal Bioanal Chem</source><year>2014</year>;
<volume>406</volume>:
<fpage>6513</fpage>–
<lpage>6528</lpage>.<pub-id pub-id-type="pmid">25173868</pub-id></mixed-citation></ref><ref id="jve1-bib-0036"><label>36.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Trypsteen</surname><given-names>W</given-names></name>,
<name name-style="western"><surname>Vynck</surname><given-names>M</given-names></name>,
<name name-style="western"><surname>De Neve</surname><given-names>J</given-names></name><etal>et al.</etal></person-group><article-title>ddpcRquant: threshold determination for single channel droplet digital PCR experiments</article-title>.
<source>Anal Bioanal Chem</source><year>2015</year>;
<volume>407</volume>:
<fpage>5827</fpage>–
<lpage>5834</lpage>.<pub-id pub-id-type="pmid">26022094</pub-id></mixed-citation></ref></ref-list><app-group><app><title id="jve1-title-0025">Appendix 1: Text mining approach</title><p id="jve1-para-0045">All articles resulting from the initial screening were searched for full-text availability through Endnote (X7) and this resulted in 2206 full-text pdfs. Next, Linux Bash commands were used for text mining:
<disp-quote id="jve1-dispquote-0001"><p id="jve1-para-0046">sfind . -name “*.pdf” -size -12M -print0 | xargs -0 pdfgrep -I -m 1 ‘droplet digital PCR | ddPCR’ 1> ddpcr_articles.txt 2> error.txt</p></disp-quote></p></app><app><title id="jve1-title-0026">Appendix 2: Table of articles reviewed</title><table-wrap id="jve1-tbl-0005" orientation="portrait" position="anchor"><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col></colgroup><thead><tr><th rowspan="2" colspan="1"></th><th rowspan="2" colspan="2">Article</th><th rowspan="2" colspan="1">Included</th><th colspan="5" rowspan="1">Exclusion criteria</th></tr><tr><th rowspan="1" colspan="1">1. Not HIV ddPCR quantification assay</th><th rowspan="1" colspan="1">2. Only HIV qPCR</th><th rowspan="1" colspan="1">3. Review</th><th rowspan="1" colspan="1">4. Data analysis method</th><th rowspan="1" colspan="1">5. Not in context of patient samples</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">Henrich</td><td rowspan="1" colspan="1">2012</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">De Spiegelaere</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">Eriksson</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">Hatano</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">Jangam</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">6</td><td rowspan="1" colspan="1">Kibirige</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">7</td><td rowspan="1" colspan="1">Massanella</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">8</td><td rowspan="1" colspan="1">Selck</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">9</td><td rowspan="1" colspan="1">Strain</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">10</td><td rowspan="1" colspan="1">Yukl</td><td rowspan="1" colspan="1">2013</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">11</td><td rowspan="1" colspan="1">Bharuthram</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">12</td><td rowspan="1" colspan="1">Beliakova-Bethe</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">13</td><td rowspan="1" colspan="1">Kiselinova</td><td rowspan="1" colspan="1">2014a</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">Kiselinova</td><td rowspan="1" colspan="1">2014b</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">15</td><td rowspan="1" colspan="1">Jones</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">16</td><td rowspan="1" colspan="1">Malatinkova</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">17</td><td rowspan="1" colspan="1">Mitchell</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">18</td><td rowspan="1" colspan="1">Rasmussen</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">19</td><td rowspan="1" colspan="1">Ruelle</td><td rowspan="1" colspan="1">2014</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">20</td><td rowspan="1" colspan="1">Bosman</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">21</td><td rowspan="1" colspan="1">de Oliveira</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">22</td><td rowspan="1" colspan="1">Janocko</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">23</td><td rowspan="1" colspan="1">King</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">24</td><td rowspan="1" colspan="1">Kiselinova</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">25</td><td rowspan="1" colspan="1">Li</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td></tr><tr><td rowspan="1" colspan="1">26</td><td rowspan="1" colspan="1">Malatinkova</td><td rowspan="1" colspan="1">2015a</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">27</td><td rowspan="1" colspan="1">Malatinkova</td><td rowspan="1" colspan="1">2015b</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">28</td><td rowspan="1" colspan="1">Mock</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">29</td><td rowspan="1" colspan="1">Pallikkuth</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">30</td><td rowspan="1" colspan="1">Perez-Santiago</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">31</td><td rowspan="1" colspan="1">Procopio</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">32</td><td rowspan="1" colspan="1">Rosadas</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">33</td><td rowspan="1" colspan="1">Ruggiero</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">34</td><td rowspan="1" colspan="1">Sogaard</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">35</td><td rowspan="1" colspan="1">Trypsteen</td><td rowspan="1" colspan="1">2015</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">36</td><td rowspan="1" colspan="1">Hong</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">37</td><td rowspan="1" colspan="1">Massanella</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">38</td><td rowspan="1" colspan="1">Sedlak</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">x</td></tr><tr><td rowspan="1" colspan="1">39</td><td rowspan="1" colspan="1">Valentini</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">40</td><td rowspan="1" colspan="1">Var</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">41</td><td rowspan="1" colspan="1">Whale</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">No</td><td rowspan="1" colspan="1">x</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">42</td><td rowspan="1" colspan="1">Kiselinova</td><td rowspan="1" colspan="1">2016</td><td rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr></tbody></table></table-wrap></app></app-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Belgique/explor/OpenAccessBelV2/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000074 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000074 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Belgique
|area= OpenAccessBelV2
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:4967968
|texte= Diagnostic utility of droplet digital PCR for HIV reservoir quantification
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:27482456" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a OpenAccessBelV2
| This area was generated with Dilib version V0.6.25. |  |